The present invention is related to process and apparatus for detecting the presence of biological agents utilizing stimulation and detection of fluorescence therefrom.
There is a recognized need for the detection of undesirable concentrations of potentially harmful airborne bacteria in health care environments, laboratories and in warfare conditions. To date, equipment used for such purposes have been unreliable and expensive to build and operate. Further, the associated support requirements have been very high, both in power requirements and maintenance. Accordingly, to date, their use has been restricted to industrial and governmental use. The service industry or the homeowner, concerned about residential and environmental monitoring, has not had practical access to such equipment.
Biological aerosols, mostly harmless, are indigenous to many environments, including homes. Microorganisms are naturally aerosolized in the atmosphere, often becoming a biological burden to downwind communities. For example, municipal wastewater aeration tanks can produce coliform bacteria, and washing of dairy processing plants can produce a persistent biological aerosol. In the hog industry, diffusion modeling has shown that aerosolized microorganisms could spread infection over a 150 km2 area among swine herds. Indoor microbiological air quality in homes and offices has demonstrated the prevalence of bacteria and fungi, for example bacterial numbers can range upwards to 800 viable particles/m3 in homes and up to 500 in offices. Typical values of fungi are about half as abundant. Fairly similar findings have bee reported as recently as 1998 by the US Environment Protection Agency which has recently taken an interest in measuring indoor and outdoor bio-logical aerosol quality.
Processes for the detection of airborne particles typically comprise sizing and characterizing the particles as biological. Such characterization of particles as being biological can accomplished by detecting a specific biomolecule present only in bio-viable particles. Most living cells contain a coenzyme or biomolecule, nicotinamide adenine dinucleotide phosphate (NADP). NADP is a biomolecule which is essential for cellular metabolism as an electron or hydrogen acceptor and is therefore an essential constituent in most biological processes to be viable. NADP is the oxidized form of NAD(P)H or nicotinamide adenine dinucleotide hydrogen (NADH). It is known that NADH can be excited with ultraviolet light and will fluoresce. The fluorescence excitation and emission wavelengths of NADH are well separated, which facilitates detection. The excitation wavelength of NADH is centered at 340 nm in the near ultraviolet spectrum, and its fluorescent emission wavelength extends from 400 to 540 nm.
Apparatus are known for implementing excitation and detection of fluorescence of NADP in discrete particles. A fluorescent aerodynamic particle sizing (FLAPS) apparatus is disclosed in U.S. Pat. Nos. 5,701,012 and 5,895,922 issued to Dr. J. Ho, the entirety of both patents being included herein by reference. The Dr. Ho disclosed the use of HeCd and tripled frequency YAG lasers, which emit a laser light beams having a wavelength in the range of 340-360 nm, ideal for the excitation of NADH. Additional aspects addressed by Dr. Ho include combining the laser excitation and detection technology with an aerodynamic particle sizer which implements converging airstreams for separating particles for analysis. Accordingly, the FLAPS apparatus comprises an aerodynamic particle sizer, the ultraviolet laser and a photomultiplier tube (PMT) for detecting fluorescence from particles. Focusing optics direct the laser to contact each passing particle. A further aspect addressed by Dr. Ho was to implement sequential lasers across the particle""s path for establishing the speed of the particle in the separating airstream, resulting in enhanced correlation with biological viability.
The lasers used in the prior art FLAPS apparatus add significantly to its complexity. For instance, the 100 cm long, 30 mW, cooled 325 nm Helium-Cadmium (HeCd) laser and focusing optics are large, heavy, and power intensive. The disclosed system weighs in at about 90 kg and consumes 800 watts of electrical power. The HeCd laser, power supply, and the particle sizing 633 nm HeNe lasers are all mounted to a 60 by 150 centimeter optics table and enclosed with a sheet metal cover to give the system a height of about 30 cm. In order to operate this apparatus remote from an industrial site requires the addition of a power generator and a technician to ensure minimal interruption between failures.
To date, the FLAPS apparatus has been limited to the use of an expensive, and unreliable, light sources such as the conventional HeCd or YAG lasers described above to provide the wavelengths suitable to excite NADH for signaling the detection of viable biomolecules and which was known to be suitable also to detect NADH even in spores.
The above prior references identify and set forth the concerns about the detection of certain hard-to-assess biological particles such as anthrax, particularly in the spore form. Questions were resolved, whether there was sufficient intrinsically fluorescing biological matter (NADH) to enable one to establish whether the particle was viable (biologically alive), and thus potentially hazardous. The 30 mW HeCd laser disclosed in the FLAPS apparatus was deemed successful for stimulating measurable fluorescence of NADH from spores.
However, as stated, the HeCd laser is expensive, unreliable and requires laser cooling and a significant power source. This severely limits its usability in critically hazardous, mobile and portable operations.
There was therefore a demonstrated need for a more practical, more portable and economical approach to biomoloecule detection. In improving the apparatus by applying a new type of laser, the applicant introduced significant uncertainly in its ability to reliably distinguish viable from non-viable particles. The laser has different operating properties and characteristics including its power output and its light wavelength; either of which could limit the apparatus""s ability to detect viable particles. This task ultimately required significant effort and expense to overcome the specific uncertainty about the ability of such improvements to provide reliable and repeatable results.
Apparatus is provided for identifying viable biomolecules in a stream. The apparatus is an improvement over related instruments, the advantages including achievement of substantial reductions in cost and power consumption which enables widening application of the technology from large industrial, governmental and military applications to also include smaller commercial and residential use. Implementation of the concepts described herein have expanded our knowledge into heretofore unknown response of biomolecules other than NADH. In the course of improving detection apparatus, the applicant has also discovered that a new range of biomolecules can be used which are indicative of particle bio-viability. Further, in expanding this range, new apparatus having improved economies and efficiencies have also been discovered while disadvantages including complexity and high power demands of the prior art apparatus are avoided.
Simply, in a preferred aspect, a laser diode is provided and applied as the excitation source. To date, laser diodes are not available at the wavelengths known to be most suited to excite the known reactive biomolecule NADH; however, they are currently available at slightly longer wavelengths. Heretofore, it has not been confirmed, whether biomolecules indicative of bio-viability exist (other than NADH) which provide similar fluorescence characteristics under laser light at other wavelengths or which are discernable at lower light emission power. It had not been confirmed whether biomolecules such as flavinoids, believed to be excited at wavelengths longer than that used to excite NADH, are even present at all in hazardous particles like spores, or are even available in sufficient quantities to be excited by laser light. Neither has it been determined that such longer wavelengths would even be suitable for exciting any biomolecules, such as flavinoids, so as to produce measurable fluorescence.
Surprisingly, applicant has determined that viable particles, including spores, can be excited to fluoresce using a laser diode operating with power output and power requirements as low as about 8-15 mWatts. Conventional apparatus using a shorter wavelength Hexe2x80x94Cd laser may also operate at relatively low power output levels, such as about 30 mW, however the practical and overall power requirements to operate the laser are many orders of magnitude greater (say 800 Watts) when the operation of necessary ancillary equipment such as laser cooling systems are included. Further, diode lasers are distinguished from conventional gas or solid state lasers by their ability to be pumped directly by an electrical current which results in efficient operation, approaching power conversion efficiencies of 50%. The gas and solid state lasers, which generally pumped by plasma excitations or an incoherent optical flash lamp source respectively, exhibit efficiencies which are more in the order of 1%. Another difference between diode and other lasers is their physical size; wherein gas and diode pumped solid state lasers are typically tens of centimeters in length, laser diode assemblies are generally about the size of the grain of salt and are only bulked up slightly (to about one centimeter) mostly for the purpose of handling and mounting purposes.
Therefore, in a broad aspect of the invention an improved apparatus is provided for identifying the existence of biologically viable particles within a particle population containing a mixture of biologically viable and biologically inert particles. The improved apparatus comprises: a solid state excitation source wherein said source is a laser diode for emitting a light beam being directed to contact particles of the particle population and having a wavelength from about 320 nm to 500 nm which is operative to excite biomolecules contained therein to produce fluorescence; a photon counter for measuring the intensity of fluorescence emitted from each contacted particle and producing a signal indicative thereof; and a microprocessor for comparing each contacted particle""s fluorescent intensity signal against predetermined criteria and establishing whether that particle is a biologically viable particle or an inert particle.
Preferably, the improvement is applied in combination with particle discretization or segregation apparatus and more preferably in combination with particle sizing apparatus for improving the identification of the biomolecules as respirable and hazardous.